In Uganda, malaria poses a considerable public health burden and the problem is exacerbated by the emergence of drug-resistant Plasmodium falciparum strains. The government interim drug policy recommends the use of the combination therapy chloroquine and sulfadoxine-pyrimethamine (CQ/SP) as the first line treatment for uncomplicated malaria. This policy has stimulated debate because CQ is no longer effective and there is increasing SP failure. The long-term objective is to provide scientific data on the molecular epidemiology of SP resistance over the next three years of CQ/SP coverage for an evidence-based re-evaluation of this policy. Plasmodium falciparum isolates in Uganda demonstrate high genotype frequencies of the dihydrofolate reductase (DHFR) triple mutant (151+R59+N108) but no detectable mutant DHFR L164 allele which is associated with very high SP resistance in South East Asia. We speculate that DHFR L164 alleles are present in Uganda at low levels undetectable by mutation-specific polymerase chain reaction since they have been detected in neighboring Tanzania. We will test the hypothesis that the DHFR L164 alleles are derived from several ancestral mutants and that the rapid emergence of a more biologically fit mutant, favored by SP selection pressure, will herald unacceptable SP failure rates and the end of SP use in Uganda. The specific aims are two-fold: to undertake annual estimates of the allele frequency of DHFR L164 in parasite isolates from a sentinel site by a high throughput dot-blot allele-specific hybridization; and to determine the evolutionary origin of DHFR L164. Community-rather than hospital-based surveys will be undertaken to determine the rate of DHFR L164 selection under SP pressure at the sentinel site over three years. The evolutionary origin of DHFR L164 will be determined by investigating polymorphic microsatellite repeats flanking the dhfr gene. The proposed approaches will facilitate the screening of at least 1,000 field isolates per year, which will greatly improve the prevalence estimates of DHFR L164 and provide insights about the parasite genetic diversity associated with the dhfr locus. The proposed work will lay the ground for a subsequent and bigger study at all 8 sentinel sites in the country. The scientific data will contribute to informed policy and consensus discussions on the continued use of CQ/SP in Uganda.